Gap junctions in the chicken pineal gland

Abstract

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.

Introduction

Gap junctions are membrane specializations that contain intercellular channels permeable to ions and small molecules allowing for synchronization of the cellular responses of heterogeneous cells. Gap junctional channels are made of a family of related proteins called connexins (Cx; reviewed in Ref. [26]). Previous studies have demonstrated the presence of gap junctions in the pineal gland of several mammalian species 11, 18, 28, 29, 34, 39, 40, 50, 56, 58, 61and teleosts [46]. Molecular characterization of pineal gap junctions has been carried out only in the rat 3, 51, where functional coupling has been demonstrated [51]. Functional coupling has also been demonstrated in the fish pineal gland [19].

Similar to other species, the chicken pineal gland secretes melatonin with a circadian rhythm. Unlike the mammalian pineal gland, the chicken pineal gland is considered a photoreceptive organ. Studies in cultures of pineal cells from 1-day-old chickens, which maintain their ability to secrete melatonin with a circadian rhythm entrained by light [15], have been widely used to elucidate components and regulation of the biological clock that governs the neuroendocrine function of the gland. These studies have revealed that individual pinealocytes are photoreceptive, and that the gland behaves as an independent oscillator 43, 55. A fine control of transcription/translation of several target genes (e.g., kinases, ion channels and regulatory elements) through circadian oscillation in the activation of transcription factors underlies the circadian rhythm of melatonin release (reviewed in Refs. 24, 33). Moreover, in the suprachiasmatic nucleus of the rat, a mammalian oscillator, astrocyte gap junctions have been proposed to facilitate synchronization of oscillation of neuronal activity [59]. Although gap junctions might play a similar role in the chicken pineal gland, these membrane specializations have not been described among the cells of this tissue.

The chicken pineal gland is composed of follicles surrounded by extracellular stroma; each follicle surrounds a small lumen. According to their ultrastructural morphology, two different cell types lie within the follicle: pineal receptor cells (B pinealocytes) and astrocytes (ependymal cells or A pinealocytes), often forming two discrete epithelial layers surrounding the lumen 6, 9. We will refer to the former cells as pinealocytes. Most cells within a follicle are elongated and stretch between the lumen at their apical pole and the basal lamina at their basal pole. Parafollicular regions contain the same cell types, perhaps in less mature forms, in less organized fashion, sometimes surrounding narrow extracellular cavities. The interfollicular stroma contains various cell types, blood vessels, lymphoid tissue, fibroblastic cells and unmyelinated nerve fibers. Numerous junctional complexes containing zonula adherens have been found in the follicular and parafollicular zones throughout development 6, 9. Neuron cell bodies are rare or absent in the chicken pineal gland, although many neurites may invade the gland from sympathetic nerves 6, 12, 62.

The circadian rhythm of melatonin synthesis and release in the pineal organ may be enhanced by synchronization of the metabolic activity of individual pineal cells, because each pinealocyte is considered an independent oscillator 43, 55. Gap junctions are good candidates for synchronizing the response of heterogeneous cell types [21]. Given that the different cell types can be distinguished morphologically much better in the pineal gland of the chicken, this organism was chosen to address the existence of coupling between homologous and heterologous cells in a system that presents a circadian rhythm in its neuroendocrine function. Thus, the experiments presented here were designed to study the presence and localization of gap junctions in the chicken pineal gland, to identify molecular components of gap junctions, and to test for intercellular gap junctional communication within the gland.